Mycobacterium tuberculosis (MTB) has been investigated in many regions, there have been only a few studies on the molecular characteristics and drug resistance phenotypes in Henan. This is the first study on the genetic profile of MTB from Henan.Tuberculosis remains a serious threat to human health as an infectious disease in China. Henan, a most populated province in China, has a high incidence of tuberculosis (TB). Though the genetic diversity of A total of 668 MTB isolates from various areas were genotyped with spoligotyping and 26-locus MIRU-VNTR . The association between TB spoligotype signatures and drug-resistant profiles was analysed.n = 558), and the typical Beijing type(ST1) was the major sublineage . In total,668 isolates were divided into 567 different types, forming 38 clusters (2–15 isolates per cluster), and 529 unique types by 26-locus MIRU-VNTR analysis. There was no correlation between the Beijing family and gender, age at diagnosis or treatment history, whereas the Beijing family was significantly associated with all four first-line drug resistance and multidrug-resistant phenotypes. For these samples, 15 of 26 MIRU-VNTR loci had high or moderate discriminatory power according to the Hunter-Gaston discriminatory index. A combination of the 10 most polymorphic loci had similar discriminatory power as the 26-locus set.Our data revealed that MTB isolates circulating in Henan had a high degree of genetic variation. The Beijing family was the most predominant genotype  contains supplementary material, which is available to authorized users. Mycobacterium tuberculosis(MTB), remains a significant public health problem worldwide. It is estimated that approximately one-third of the world’s population has been infected with MTB, and 1.8 million people die of this disease annually. Among the 22 high TB burden countries reported by the World Health Organization, China ranks second in the world with approximately 1.3 million new cases [Tuberculosis (TB), caused by Recently, molecular epidemiology tools have been used to assess risk factors associated with recent transmissions ,to trackNon-coding regions of the MTB genome contain a set of identical 36-bp direct repeats (DRs), which are separated by 35- to 41-bp unique DNA spacer sequences. Spoligotyping, a rapid and highly reproducible method, detects the presence or absence of DR loci . The resMIRU-VNTR, a new PCR-based typing method, determines the size and repeated number of units in each locus by amplifying mycobacterial interspersed repetitive units. Easy operation, economical cost, reproducible results and high discriminatory power make it practical for routine use , and theM.tuberculosis transmission model can help determine risk factors and improve contact tracing. Moreover, little was known about the genetic diversity of MTB in this region until now. The study is the first to use26-locus MIRU-VNTR, including the standard 24-locus and two other loci (ETRF and Mtub38) [In China, more than 80% of tuberculosis patients are in rural communities . Henan P Mtub38) , 22},forM. tuberculosis H37Rv was used as the control strain.In total, 668 strains of MTB were collected from smear-positive pulmonary TB patients in the Tuberculosis Control Institution from various regions of Henan Province during 2015. The patients were from the following regions: 70 from Zhengzhou, 70 from NanYang, 65 from Zhoukou, 50 from Zhumadian, 50 from Luoyang,50 from Kaifeng, 50 from Shangqiu, 45 from Xinyang, 40 from Xinxiang, 35 from Pingdingshan, 35 from Anyang, 20 from Puyang, 19 from Luohe, 18 from Xuchang,18 from Jiaozuo,18 from Sanmenxia, 15 from Hebi, and none from Jiyuan. This project was approved by the Ethics Review Committee of Henan CDC. All the patients with pulmonary TB provided informed consent before participation in this investigation. Ethics were respected throughout the whole study period.g for 5 min. The supernatant containing DNA was used as a template for PCR.All the isolates were cultured in Lowenstein-Jensen (L-J) medium for 3–4 weeks at 37 °C. A loopful of colonies was added to 300 μl of TE buffer at pH 8.0. The suspension of bacterial cells was incubated at 85 °C for 30 min to inactivate pathogens, followed by centrifugation at 13,000 These collected isolates were subjected to spoligotyping on commercially available membranes according to a previously described standard protocol –25. BrieMIRU-VNTR typing based on 26 loci, including the standard 24 loci  and 2 otEpidemiological information, such as age, sex, and clinical treatment history of the patients, was collected with a questionnaire method.The sputum samples of clinical patients were isolatedand cultured using Lowenstein-Jensen (L-J) media. Then, the drug susceptibility tofirst- and second-line anti-TB drugs was determined using the proportion method with H37Rv as a control . The drup ≤ 0.05) was considered statistically significant.The Hunter-Gaston discriminatory Index (HGDI) was calculated as previously described to assess allelic diversity at VNTR loci . The datIn this study, 668 isolates were clustered into 35 distinct genotypes by spoligotyping. The octal coded spoligotyping results were assigned spoligotyping shared international type (SIT) numbers and then compared with those in spolDB4  and theuTo evaluate the genetic relations among all 668 isolates, the minimum spanning tree was generated using BioNumerics 6.6 software. Each node represents a distinct genotype, and the size of each node depends on the number of the corresponding cluster . These iThe analysis of 26-locus MIRU-VNTR showed that 139 isolates (23.9%) grouped into 38 clusters, and the remaining 529 isolates shared unique patterns. The largest cluster contained 15 strains, and the other clusters consisted of 2–14 isolates. The clustering rate of 26-locus MIRU-VNTR was 15.1%. There was some discrepancy between the spoligotyping and 26-locus MIRU-VNTR for the Beijing family isolates. The largest cluster of the MIRU-VNTR mainly included Beijing family isolates; however, 33 isolates belonged to the non-Beijing family patterns in this largest clade  for each locus. As previously reported, MIRU-VNTR loci were further designated highly (> 0.6), moderately (0.3–0.6), or poorly (< 0.3) discriminatory loci according to the HGDI scores . These lWe further compared the allelic diversity between the Beijing family and the total isolates. All 26 loci showed lower allelic diversity among the Beijing family isolates, which was in accordance with the closer affinity of this genotype , and the Beijing family also showed a higher risk for developing drug resistance to all four first-line drugs .There was no apparent difference in the proportions of other drug-resistant profiles between the Beijing and non-Beijing family genotypes  were resistant to at least one drug, 50 (7.5%) isolates were classified as MDR-TB, and 30 isolates (4.5%) were susceptible to all four first-line drugs. As shown in Table Then, the sociodemographic characteristics of patients, including gender, age at diagnosis, and clinical history, were compared between the Beijing and non-Beijing family genotypes, and no statistically significant differences were detected Table .Table 6EM. tuberculosis in this region. This is the first study to investigate the allelic diversity of MTB isolates in Henan Province using spoligotyping and MIRU-VNTR.The Beijing family genotype remains the predominant genotype in China ; howeverM. tuberculosis is divided into 162 clades according to the international spoligotyping database SpolDB4 [ SpolDB4 , and the SpolDB4 . The pat SpolDB4 , 33, Tia SpolDB4 , Tibet  belonged to the Beijing family, three to the T1 family, one to MANU2, and one to the H3 family. Moreover, 10 (34.5%) of these 29 isolates were resistant to one or more drugs. However, it remains uncertain whether the patients infected with these strains had close contact with each other. Since the ability of the different locus sets to classify MTB was diverse in Henan, we needed to determine an optimal set that had a discriminatory power comparable to that of the 26-locus set. In consideration of labour and economic costs, we chose the top 10 loci combination, which had an HGDI value comparable to that of the classical 24-locus set (0.996 vs. 0.997), which was slightly lower than that of the 26-locus set (0.998). These data indicated that the 10-locus combination was useful and cost-efficient. Therefore, we suggest this 10-locus set as a potential first-line MTB genotyping method in Henan Province, especially for a large-scale molecular epidemiological survey.Our data revealed some inconsistency between the results of spoligotyping and MIRU-VNTR for several MTB isolates. Beijing family isolates comprised a large proportion of the largest cluster by MIRU-VNTR genotyping; however,33 isolates with a non-Beijing spoligotype were found in this largest clade. Furthermore, another 19 strains showed the Beijing genotype spoligotype pattern but could not be distinguished by MIRU-VNTR  UPGMA dendrogram generated by the 26-locus MIRU-VNTR, (2) spoligotyping patterns, and (3) strain number. (PDF 113 kb)


Europium (Eu)-doped fluorapatite (FA) nanorods have a biocompatibility similar to that of hydroxyapatite (HA) for use as cell imaging biomaterials due to their luminescent property. Here, we discuss the new application of europium-doped fluorapatite (Eu-FA) nanorods as an anticancer drug carrier. The Eu-FA nanorods were prepared by using a hydrothermal method. The morphology, crystal structure, fluorescence, and composition were investigated. The specific crystal structure enables the effective loading of drug molecules. Doxorubicin (DOX), which was used as a model anticancer drug, effectively loaded onto the surface of the nanorods. The DOX release was pH-dependent and occurred more rapidly at pH 5.5 than at pH 7.4. The intracellular penetration of the DOX-loaded Eu-FA nanorods (Eu-FA/DOX) can be imaged in situ due to the self-fluorescence property. Treatment of melanoma A375 cells with Eu-FA/DOX elicited a more effective apoptosis rate than direct DOX treatment. Overall, Eu-FA exhibits potential for tracking and treating tumors and may be potentially useful as a multifunctional carrier system to effectively load and sustainably deliver drugs. In comparison to fluorescent dyes or semiconductor quantum dots (QDs), these lanthanide-doped nanoparticles are advantageous due to their photostability, sharp visible emission bandwidth, and nontoxicity [Fluorapatite (FA) is one of the inorganic constituents of bone or teeth and is used for hard tissue repairs and replacements . FA(Ca10 F− ions . However10PO6F2) can  studies ,6. Some toxicity ,8,9. In toxicity .Their high surface-to-volume ratio, reactivities, and biocompatibility make FA nanorods favorable for use in drug delivery studies ,13. In t5D0-7F1) nm and 617 nm (5D0-7F2) upon excitation at 405 nm. Doping of Eu3+ ions substitute part of the Ca2+ ions in the Eu-FA crystals. The proposed lattice models , so it can easily inter-convert from one functional group to other by changing the pH value of the solution [The loaded DOX was subsequently released into PBS with different pH values. Although a pH of 7.4 is representative of normal physiological conditions, an acidic pH of 5.5 is shared by the extracellular tissues of tumor cells . The DOXolecules . The DOXsolution ,20. It iNext, the cellular uptake of Eu-FA was examined. The A375 cells were treated with 10 μg of Eu-FA nanorods for 4 h. The intracellular penetration of Eu-FA/DOX can be imaged in situ due to the self-fluorescence property. The location of the Eu-FA nanorods in the A375 cells was confirmed by confocal laser scanning microscopy. The red regions represent the fluorescence signal of Eu-FA, and the blue signals arise from the DAPI-stained nucleus. The red fluorescence signals of Eu-FA were observed in the cytoplasm  or within the nucleus  of the A375 cells treated with DOX B. TherefTo investigate the apoptosis rate induced by Eu-FA/DOX, the treated cells were double-stained with FITC-Annexin V and PI followed by analysis with flow cytometry. Propidium iodide (PI) was used in conjunction with Annexin V to determine if the cells were viable, apoptotic, or necrotic from differences in the plasma membrane integrity and permeability. This approach is a universal and accurate method for detecting cell apoptosis . After a3+-doped FHA nanoparticles were synthesized via a hydrothermal method . All chemicals were analytical grade. Exactly 0.5 g of octadecylamine were dissolved in 4 mL of oleic acid while heating, and 16 mL of ethanol and an aqueous solution of Ca(NO3)2  were then added while stirring. NaF , Eu(NO3)3 , and Na3PO4  were added to the solution followed by an additional 5 min of stirring. Then, the sealed flask was heated at 160 °C for 16 h. The obtained mixture consisted of FA nanoparticles doped with Eu3+, and these nanoparticles were collected by centrifugation and freeze-dried.EuTransmission electron microscopy (TEM) was carried out on a JEM-2100 instrument to determine the sizes and morphologies of the Eu-FHA nanorods. The samples were prepared by placing a drop of a dilute ethanol dispersion of the products on the surface of a copper grid. XRD analysis was performed using an Image Plate X-ray Diffractometer  with Cu Ka radiation. The luminescence spectra were recorded on a Hitachi F-4500 fluorescence spectrophotometer. Luminescent photography of the sample was performed under UV light at 390 nm. The DOX loading of Eu-FA nanorods were prepared using an absorption method. Doxorubicin hydrochloride  was dissolved in the PBS solution at pH 7.4 to produce stock solutions with concentrations of 0, 200, 750, 2000, 3000, 4000, and 5000 μg/mL. Five milligrams of the Eu-FA sample were ultrasonically dispersed in each DOX solution for 5 min, and then kept in a 37 °C water bath for 12 h. To quantify the drug-loading amount, the nanorods were centrifuged at 10,000 rpm for 5 min, and the supernatant was gathered and detected by UV-vis spectrophotometer at an absorbance of 483 nm.To study the release curve of DOX from the nanocarrier samples, the as-prepared Eu-FA/DOX nanorods were used. A 2 mg sample was dispersed in a 2 mL PBS solution , followed by dialysis  in an air bath oscillator at 37 °C. The dialysis bag was placed in a buffer medium of 8 mL, and the vibration was slow under the condition of 37 °C. At the different time points , the amount of drug released into the medium was measured by using a UV-Vis spectrophotometer with an absorbance of 483 nm, and the release rate was then calculated. 5 cells in each well of 6-well culture plates and treated with Eu-FA nanorods at a concentration of 10 μg/mL for 4 h. The fluorescent images were observed and analyzed using a laser scanning confocal microscope . The cells were counterstained with DAPI  to observe the nucleus.Melanoma A375 cells were seeded at 10The A375 cells treated with Eu-FA, DOX, or Eu-FA/DOX for 24 h were harvested. The concentration of the Eu-FA was 40 μg/mL, the DOX concentration was 17.48 μg/mL, which was calculated by the maximum drug loaded amount at the adsorption value of 43.7%, and the initial pH value of the solution was 7.4. Flow cytometry was employed to detect cell death and apoptosis according to the manufacturer’s instruction. After being harvested via trypsinization and lysed with ice-cold PBS, the cocultured cells with Annexin V-FITC  were incubated for 15 min in a dark at room temperature. Then, the re-suspended cells were stained with propidium iodide . A Becton Dickinson FACS/Calibur cytometer  was employed to analyze the Annexin V-FITC and propidium iodide signals.t-tests were performed to determine the statistical significance between experimental groups. p < 0.05 was considered to indicate statistical significance. All results are expressed as the mean ± standard.Statistical analysis was performed using the SPSS 22.0 software. Student’s In summary, the as-synthesized Eu-FA nanorods exhibited excellent photoluminescence and high drug-loading capacity. We believe that this multifunctional nanocarrier will generate further interest and open new avenues for research on the applicability of using these nanoparticles in cancer treatment.


Entamoeba histolytica which allows the microbe to preserve its virulence factors and ability to induce hepatic abscesses in animal models. The novel CLUPS medium is an improved version of the PEHPS medium, previously designed in our laboratory. The main difference is the substitution of raw beef liver in PEHPS by raw beef lung in the CLUPS medium. To compare the performance of three-culture media , E. histolytica trophozoites were cultured in quintuplicate, followed by the evaluation of phospholipase activity and the induction of liver abscesses in golden hamsters. E. histolytica trophozoites grew significantly better in CLUPS medium than in TYI-S-33. Likewise, CLUPS-cultured trophozoites produced significantly more phospholipases than TYI-S-33-cultured trophozoites. Finally, trophozoites grown in any of the three tested media had similar potential to induce liver abscesses.Amebiasis remains a major health problem in Mexico. Therefore, the search for better culture media and low-cost diagnostic and therapeutic tools is fundamental. We present a new culture medium for Amebiasis is one of the main causes of infectious disease in Mexico and African countries. The World Health Organization reported in 1997 that there were about 40 million disease cases worldwide per year and estimated that over 400 million people were asymptomatically infected . Unfortu Entamoeba species have been recognized [ Entamoeba histolytica remains a major cause of gastrointestinal disease in Mexico and other parts of the world [ E. histolytica is present in 16-20% of people with a gastrointestinal disorder [ in vitro study of microorganisms such as E. histolytica. Ideally, cultured parasites should present the same growth and virulence characteristics as their counterparts in their natural habitat [ E. histolytica was first accomplished by Diamond in 1961 [ E. histolytica axenic culture media are a source of peptides, amino acids (trypticase or casein digest peptone), nucleic acids (yeast extract), carbohydrates (glucose), lipids (serum), and vitamins. The development of a new culture medium for intestinal protozoan allows learning about the biology of parasites, their growth rate, virulence factors (like phospholipase activity), susceptibility to new drugs, and the appearance of resistant strains, among other aspects [On the other hand, since the start of this century, novel nonpathogeniccognized , 4. Ongocognized . The inche world , 7. PCR- habitat . Axenic  in 1961  that rec in 1961 . This ne in 1961 . A casei in 1961 , 13. In  in 1961 . PHEPS i in 1961 . The mai aspects . in vitro growth of E. histolytica [The PEHPS medium was designed because of the need of an economic medium without the interlot variability present in manufactured culture media. In the commercial media TYI-S-33 and TPS, the supplements, Panmede, and yeast extract present batch-to-batch variability , whereastolytica . E. histolytica growth without affecting virulence factors nor the ability to cause hepatic abscesses in animal models.The main objective of this study was to demonstrate that the new CLUPS medium facilitates properBeef liver, lungs, and pancreas and pork pancreas were obtained at a local slaughterhouse from recently sacrificed animals. All organs were transported in ice and manipulated as previously described . Extractμm filter  and stored in 5.5-mL aliquots at -20°C. Just before use, CLUPS is supplemented with 8.3% v/v bovine serum. Bovine serum was also produced in-house as follows: about 15 L fresh blood from cows for human consumption was collected in a sterile way at a local slaughterhouse. After blood coagulation, the serum was recovered, and the complement was heat-inactivated . Next, the serum was prefiltered through Whatman No. 1 paper before being sterile-filtered through a series of sterile Millipore HAWP filters  from 5 to 0.22 μm. Serum was frozen at -70°C until use.The composition and preparation of CLUPS medium are the same as those of PEHPS medium , except  E. histolytica HM1: IMSS strain were cultured in TYI-S-33 medium under axenic conditions. The reference strain was maintained in 13 × 100 mm borosilicate screw-capped tubes containing 5.5 mL TYI-S-33 medium plus 0.5 mL bovine serum. Each tube was inoculated with 1 × 104 trophozoites/mL and incubated at 36.5°C for 72 h until the amebas were harvested as described [Trophozoites from theescribed . Ameba described  before bFor assays, the parasites were maintained in axenic conditions at 36.5°C for 30 days by serial subcultivation in TYI-S-33, PEHPS, or CLUPS medium. E. histolytica in TYI-S-33, PEHPS, or CLUPS medium were determined in quintuplicate every 24 h for 5 days. A hemocytometer was used to determine the number of trophozoites/mL, and the doubling time was calculated by linear regression [Growth curves ofgression . The yie x g, 10 min). The protozoa was resuspended and diluted in 2 volumes of Hank's balanced salt solution.Next, the trophozoites were homogenized using a motor-driven Elvehjem-Potter Teflon/glass homogenizer (100 strokes) to obtain the “total extract”. The “total extract” was centrifuged at 30,000 x g for 10 min. Both the supernatant (S30) and the precipitate (P30) were stored in 200-μl aliquots at -70°C until use.Phospholipase A activity can be recovered from fraction P30 using a previously described cellular fractionation method . Culture1 and A2 was determined according to Vargas-Villarreal et al. [14C)-phosphatidylcholine (14C-PC). Radioactivity in the chromatographic spots corresponding to 14C-LPC, but not in those of 14C-FFA, indicates type A1 activity; in the opposite case, there would be type A2 phospholipase activity. Radioactive substrates were purchased from New England Nuclear, Boston, Mass. Each of the above (4 μCi per assay) was mixed with 1.0 mL 0.1 M sodium Tris-base buffer (pH 8.0), 2 mM CaCl2, 0.2% Triton X-100, and 0.27 mM phosphatidylcholine. The mixtures were sonicated with an Ultratrip Labsonic System , which was operated at 40 W for 60 s. The resultant emulsion was distributed in 0.5-mL aliquots and stored at –70°C until use.The phospholipase activity of types Al et al. . The assμL containing 200 μg total proteins) was mixed with 10 μL substrate and incubated at 36.5°C (water bath) for 2.5 h. The reaction was stopped by adding 25 μL 5% trichloroacetic acid/n-butanol stop solution containing the following internal controls: rat liver free fatty acids , egg-yolk lysophosphatidylcholine , and egg-yolk phosphatidylcholine .The P30 fraction (10 14C-PA)-PC.The hydrolysis mixtures were located drop-by-drop on the origins of 10 × 10 cm silica-gel thin-layer chromatography plates . The plates were placed into a developing tank with a solvent system composed of chloroform:methanol:acetic acid:water (140:40:16:8 volumes). The lipid spots were developed by exposure to iodine vapor . The spoProtein concentrations were quantified by the Lowry method .Mesocricetus auratus), weighing 40-60 g [6 trophozoites grown in PEHPS, CLUPS, or TYI-S-33 medium (n=4). The negative control group was inoculated with parasite-free medium. After inoculation, the incision was sutured using a continuous padlock [ ad libitum for three days, until they were sacrificed by euthanasia with an anesthetic overdose . After making sure that the animal had died (No breathing or heartbeat), an exploratory laparotomy was performed to evaluate liver abscess formation by direct observation and measurement of the lesion. Here to, fine cuts were made in the liberated livers of all animals with a sterile scalpel blade until liver lesions were found and could be measured.All animal work realized in this research was realized according to international ethical guidelines. All procedures were performed by a veterinarian expert in animal handling. Freshly weaned, male golden hamsters ; the doubling time in PEHPS was 22.0 h and in TYI-S-33 23.2 h.After an adaptation lag, trophozoites ofTYI-S-33 . Trophoz14C-PA)-LPC and (14C) palmitic acid accumulated as a function of incubation time; the increase was almost linear the first 60 min, then slowed down, and reached a plateau at 90 min. Trophozoites grown in any of the three-culture media maintained the production of phospholipases A1 and A2 -PC increases linearly as a function of P30 protein amount in both A1 and A2 phospholipase activity  for the axenic growth ofS medium ; the rawS medium . E. histolytica HM-1 strain in continuous culture with yeast-extract-free PEHPS for over 25 years [At 72 h of cultivation, the trophozoite yield in CLUPS was 30% higher than in TYI-S-33. This superior performance was significant and cannot be explained by simple media variability . When co25 years , 21. TheWhen we compared the growth pattern between CLUPS and PEHPS, it seems that trophozoite growth starts and ends earlier in CLUPS than in PEHPS. This retardation in PEHPS may be due to the presence of antiparasitic or antibiotic residues in the raw beef liver –20, whic E. histolytica trophozoites; however this would have to be demonstrated by prolonged use.The production of phospholipases is the same for both media; the seemingly higher production in CLUPS was not statistically significant. So far, it seems that the CLUPS medium outperforms PEHPS medium for the culture of in vivo by the formation of hepatic abscesses; it has even been shown that amebas increase their virulence after inoculating them in hamster liver and recovering them from lesions [The effectiveness of virulence factors in amebas is demonstrated lesions . Althoug lesions . We carrAlternatively, the CLUPS medium may contain factors that favor proper growth and an increased phospholipase activity. To verify if lung extracts have better factors than the other culture media, we will compare the growth and enzymatic activity of amebas cultivated in either CLUPS or PEHPS medium. At the moment no commercial medium has extracts from animal organs , 11.The successful culture of this parasite in this new CLUPS medium allows for the execution of assays in an economical and stable environment. Similar to the PEHPS medium, CLUPS avoids assay variability due to interlot variability common in commercial culture media . In this E. histolytica in CLUPS medium allows us to investigate the biology of this parasite, to study its virulence factors, to develop better diagnostic methods, and to test new drugs for better treatments.The reliable cultivation ofIn conclusion, we consider that the CLUPS medium is a superior alternative culture medium, which is both economic and free of the interlot variability present in commercial TYI-S-33.


The aim of this study was to test the potential of a new elastin-specific molecular agent for the performance of contrast-enhanced first-pass and 3D magnetic resonance angiography (MRA), compared to a clinically used extravascular contrast agent (gadobutrol) and based on clinical MR sequences.  Eight C57BL/6J mice  underwent a contrast-enhanced first-pass and 3D MR angiography (MRA) of the aorta and its main branches. All examinations were on a clinical 3 Tesla MR system . The clinical dose of 0.1 mmol/kg was administered in both probes. First, a time-resolved MRA (TWIST) was acquired during the first-pass to assess the arrival and washout of the contrast agent bolus. Subsequently, a high-resolution 3D MRA sequence (3D T1 FLASH) was acquired. Signal-to-noise ratios (SNRs) and contrast-to-noise ratios (CNRs) were calculated for all sequences. p > 0.05). Following the bolus phase, both agents showed a comparable intravascular enhancement . Both agents resulted in a high image quality with no statistical difference (p > 0.05).  The elastin-specific MR probe and the extravascular imaging agent (gadobutrol) enable high-quality MR angiograms in all animals. During the first-pass, the probes demonstrated a comparable peak enhancement (300.6 ± 32.9 vs. 288.5 ± 33.1,  The novel elastin-specific molecular probe enables the performance of first-pass and late 3D MR angiography with an intravascular contrast enhancement and image quality comparable to a clinically used extravascular contrast agent. Contrast-enhanced magnetic resonance angiography (CE-MRA) has become the clinical reference technique for the evaluation of vascular territories and of associated pathologies in patients , 2. CE-MRecently, a novel small-molecular-weight gadolinium-based elastin-specific molecular agent has been introduced –8. This The aim of this study was to test the potential of a small-molecular-weight gadolinium-based elastin-specific magnetic resonance (MR) probe for the performance of contrast-enhanced first-pass and late 3D MRA using clinical MR protocols in comparison to a clinically used extravascular contrast agent (gadobutrol).µg/kg), fentanyl (50 µg/kg), and midazolam (5 mg/kg). Following the final imaging session, mice were euthanized. For histological examinations, a perfusion at a pressure of 100 mm Hg with the fixative MorFFFix®  was performed followed by excision of the carotid arteries, brachiocephalic artery, and aorta including the renal arteries. All animal procedures in this study were conducted by a veterinarian and all possible steps were taken to avoid animal suffering at each stage of the experiment.All procedures were performed according to the guidelines and regulations of the Federation of Laboratory Animal Science Associations (FELASA) and the local guidelines and provisions for the implementation of the Animal Welfare Act. For imaging, eight ten-week-old male homozygous C57BL/6J mice from Charles River Laboratories  were used. All animals were housed in a clean barrier and fed with a standard lab diet. Prior to the imaging sessions, mice were anesthetized using an intraperitoneal administration of a combination of medetomidine  and a clinical single-loop coil . During the imaging sessions, body temperature (37°C) was monitored using a MR-compatible heating system . For the administration of the different MR imaging agents, a small diameter tube with an attached needle was inserted into the tail vein of the animals. Following the acquisition of unenhanced scans, the administration of the imaging agents was performed.R1 relaxivity of gadobutrol at 3T was reported to be 3.6 ± 0.2 [As a “control” agent, gadobutrol  was administered undiluted at a clinical dose of 0.1 mmol/kg bodyweight and followed by 0.01 ml saline. In a previous study, the .6 ± 0.2 . The ela.6 ± 0.2 . The imaAll imaging sequences used in this study were derived from a clinically used MR protocol and adapted for small animal imaging. For image acquisition the following sequence parameters were used. First, a low-resolution localizer scan was performed in sagittal, coronal, and transverse orientation to plan the subsequent imaging planes. The localizer scan had the following parameters: field of view 280 mm, matrix 256, and slice thickness 6 mm. Subsequently, a TWIST (time-resolved angiography with interleaved stochastic trajectories) T1 angiography sequence was performed: Repetition time (TR) 3.5 ms, echo time (TE) 1.3 ms 10 slices, flip angle 12°, 50 timepoints, field of view 316, matrix 480, interpolated inplane resolution 0.3 mm, and slice thickness 0.5 mm. For the late-phase high-resolution angiography, a 3D T1 FLASH (fast low angle shot) sequence was used: TR 4.7 ms, TE 1.9 ms, 60 slices, field of view 316, matrix 480, interpolated inplane resolution 0.3 mm, and slice thickness 0.5 mm.MR images were analyzed using OsiriX . All images were analyzed in a random order and blinded to the imaging agent and the different time points. Signal-to-noise (SNR) and contrast-to-noise (CNR) measurements were performed in data sets of the time-resolved MR angiography (TWIST) and high-resolution 3D FLASH imaging. To quantify the vascular signal enhancement in the time-resolved TWIST sequence, signal measurements were performed using a region of interest in the left ventricle of the heart. To quantify the vascular signal enhancement in the T1 FLASH sequence, signal measurements were performed using a region of interest in the aortic lumen at the level of the heart. Noise measurements were performed anterior to the thorax with the standard deviation of the region of interest. The SNR was calculated as follows: SNR = mean signal (aortic lumen)/standard deviation signal (noise). For the assessment of the contrast-to-noise ratio, an additional region of interest was placed in the muscle tissue dorsal of the aorta. The CNR was calculated as follows: CNR =  − mean signal (muscle tissue))/standard deviation signal (noise).For the image quality assessment of the 3D T1 FLASH sequence, the image quality was ranked based on a five-point scale: 1 = poor quality information, 2 = aorta visible but markedly blood, 3 = aorta visible with moderate blurring, 4 = aorta visible with minimal blurring, and 5 = aorta visible with sharply defined borders. The image grading system used was modified based on the system by McConnell et al. .µm thick serial sections. These sections were subsequently stained with Miller's Elastica van Gieson stain (EvG) and hematoxylin and eosin (HE).The histological analysis was performed for the aorta, the brachiocephalic artery, and the carotid artery. The vessels were embedded in paraffin and cut into 5 -in vivo MR images and ex vivo images from histology. The morphometrical analysis was performed using elastin-stained sections (Miller's Elastica van Gieson stain) and ImageJ software .The complete arterial system including the carotid arteries, the subclavian arteries, brachiocephalic artery, aortic arch, and descending aorta to the iliac arteries was visualized in all MR scans. The vessel bifurcations, e.g., aorta to brachiocephalic artery, brachiocephalic artery to subclavian artery, were used as anatomical landmarks for coregistration of the t-test  was used. Linear regression analysis and Bland-Altman analysis were used to compare the two contrast agents. For the first method, the slope of the regression line (b) together with the Pearson correlation coefficient (R) was calculated. For the latter method instead, the mean difference (M) with limits of agreement (I) (±1.96 times the standard deviation) was calculated. p < 0.05 was used for significance.Data are expressed as mean ± standard deviation. To determine continuous variables Student's No side effects or adverse reactions to the imaging agents were observed in the investigated animals.p > 0.05; CNR: 65.3 ± 7.5 vs. 61.4 ± 7.7, p > 0.05). For the peak enhancement, the average aortic SNR and CNR was not significantly different between the elastin-specific agent and the extracellular extravascular agent gadobutrol (p > 0.05). Also, in the later phases after more than 20 seconds, the average SNR and CNR between the contrast agents was not significantly different from each other (p > 0.05) for each time point measured.An example of the dynamic contrast enhancement using the elastin-specific molecular agent and gadobutrol is shown in Figures p > 0.05) and the CNR (p > 0.05).A typical high-resolution 3D T1 FLASH angiography is shown in Figures p ≤ 0.05).The image quality for both the elastin-specific molecular agent and the extracellular extravascular agent gadobutrol were high for the visualization of the thoracic and the abdominal aorta . For theIn vivo and ex vivo luminal area measurements were performed in the high-resolution 3D angiography (T1 FLASH) following the administration of the elastin-specific molecular agent and the extracellular extravascular agent gadobutrol . All measurements were performed at the same clinical dose of both probes and at a clinical field strength with a clinical sequence design.158Gd-labelled C32H40N7O11  elastin-specific molecular probe is a low-molecular-weight contrast agent. Comparable to clinically used gadolinium-based contrast agents, the relatively small probe is labelled with a single gadolinium chelate [via the hepatobiliary system. By contrast, the high measured probe concentration in kidney and urine indicated the rapid renal clearance of the agent. Overall, these properties represent a favourable pharmacokinetic profile of the elastin-specific molecular probe comparable to clinically used gadolinium-based contrast agents, such as gadobutrol.The paramagnetic For the visualization of the vascular system in patients and the characterization of pathologies, different types of MR probes, including gadolinium-based and iron oxide-based agents have been investigated , 11. In In this study, we investigated the properties of a novel elastin-specific gadolinium-based molecular probe for the visualization of the clinically important early arterial phases. Importantly, all measurements were performed at the same clinically relevant dose (0.1 mmol/kg) with a single bolus administration. Additionally, all imaging was performed with clinically used MR sequences and at a clinical field strength of 3T. The elastin-specific gadolinium-based molecular probe demonstrated comparable imaging properties to the routinely used clinical contrast agent gadobutrol for the performance of an MR angiography. Following the intravenous injection, both agents were shown to demonstrate a comparable rapid renal clearance from the blood pool. Because of this property, the highest intravascular signal could be achieved during the first-pass following the intravenous administration. Additionally, both agents showed a similar SNR and CNR during the first-pass and a comparable washout pattern, which is in line with previous studies –8. The SIn summary, this study demonstrated that the elastin-specific molecular probe shows excellent signal properties for the acquisition of time-resolved first-pass and late 3D MR angiographies. Signal enhancement of the aorta was comparable to what was measured with the clinically used agent (gadobutrol) at the same dose. This indicates that first-pass angiography and tissue perfusion, e.g., for tumor evaluation could be performed with comparable SNR and CNR as with currently used clinical gadolinium-based agents. The main advantage of the elastin-specific probe, compared to unspecific clinically used probes, is that it can also be used for the visualization of specific pathological processes with the expression of (tropo-)elastin in delayed imaging.This study has several advantages regarding the translation of the results into clinical applications: (1) All imaging were performed at a clinically relevant field strength, which allows direct translation to human applications. (2) The MR sequences used in this study are based on a clinical MR imaging protocol. (3) The molecular character and size of the used probe is directly comparable to clinically approved contrast agents, increasing the probability of a translation into clinical applications. Despite the prevalence of high-field scanners in previously published small animals MRI studies, clinical MRI systems are being increasingly used for preclinical research. This study was performed on a clinical 3T MR imaging system for several reasons. The effects on T1 and T2 relaxation, rotational correlation and signal properties of molecular probes, and potential sources of artefacts  can vary substantially depending on the applied field strength (1.5–3T vs. >7T) and may therefore limit the direct translation of the findings to human application . AdditioThis study has several limitations. The MR probe was evaluated in an experimental small animal study. However, previous experimental studies have shown that this type of study can yield a good indication of how a MR agent could perform in a clinical setting. Secondly, a relatively small number of animals with defined doses of the MR agent was used. Additionally, a single acquisition protocol with a single regime of contrast injection was applied. This acquisition protocol was directly derived from the clinical imaging protocol and scaled down for this study. Imaging was performed only at 3T and therefore the results are not directly applicable to 1.5T scanners.The novel elastin-specific molecular probe enables the performance of a contrast-enhanced first-pass and late 3D MR angiography with an intravascular contrast enhancement and image quality comparable to a clinically used extravascular contrast agent.


Verticillium dahliae, is the most devastating soil-borne fungal disease of olive trees, and leads to low yields and high rates of tree mortality in highly susceptible cultivars. The disease is widely distributed throughout the Mediterranean olive-growing region and is one of the major limiting factors of olive oil production. Other than effects on crop yield, little is known about the effect of the disease on the content of volatile compounds and phenolics that are produced during the oil extraction process and determine virgin olive oil (VOO) quality and commercial value. Here, we aim to study the effect of Verticillium wilt of the olive tree on the content of phenolic and volatile compounds related to the sensory properties of VOO. Results showed that synthesis of six and five straight-chain carbon volatile compounds were higher and lower, respectively, in oils extracted from infected trees. Pathogen infection affected volatile compounds known to be contributors to VOO aroma: average content of one of the main positive contributors to VOO aroma, (E)-hex-2-enal, was 38% higher in oils extracted from infected trees, whereas average content of the main unpleasant volatile compound, pent-1-en-3-one, was almost 50% lower. In contrast, there was a clear effect of pathogen infection on the content of compounds responsible for VOO taste, where average content of the main bitterness contributor, oleuropein aglycone, was 18% lower in oil extracted from infected plants, and content of oleocanthal, the main contributor to pungency, was 26% lower. We believe this is the first evidence of the effect of Verticillium wilt infection of olive trees on volatile compounds and phenolics that are responsible of the aroma, taste, and commercial value of VOO.Verticillium wilt, caused by the defoliating pathotype of  Verticillium dahliae, is the most devastating soil-borne fungal disease of olive trees (Olea europaea L.). The disease is widely distributed in all Mediterranean olive-growing regions, and is one of the major limiting factors of olive oil production  are key characteristics that distinguish it from other culinary oils, and given that VOO is the only food product to undergo sensory analysis for classification .Most volatile compounds present in VOO are six straight-chain carbons (C6) aldehydes and alcohols, and their corresponding esters, produced by enzymes of the lipoxygenase (LOX) pathway . The actp-HPEA) and its hydroxyl derivative, hydroxytyrosol , and are mainly the dialdehydic forms of decarboxymethyloleuropein and decarboxymethylligstroside aglucones  and aldehydic forms of oleuropein and ligstroside aglucones   is understood to be the principal phenolic responsible for VOO pungency  . In addictively) , p-HPEA-pungency , while 3pungency .Verticillium wilt is one of the major limiting factors of olive oil production and other than effects on crop yield, little is known about the effect of the disease on VOO quality and commercial value. In the context of the relationship between volatile and phenolic compounds and sensory characteristics in VOO , we aim V. dahliae isolate V138I from defoliated Coker 310 cotton plants from Córdoba (Spain) as a representative of the defoliating (D) pathotype in an inoculum that consisted of an infested cornmeal-sand mixture   were selected as representative of the different soil types commonly found in Andalusia, southern Spain, the main olive growing area in the world with more that 1.5 million hectares, producing more than one-third of the world’s olive oil . Since w:2, w/w) . The inoV. dahliae. In March, we transplanted single, 1-m tall, 18-month-old “Picual” plants to 40-L pots containing one of the three types of infested or non-infested soils. Pots were buried in a field plot at the Institute of Sustainable Agriculture, Córdoba, Spain, to maintain soil temperature at a level similar to that in field soil, and plants were grown under standard drip irrigation and fertilization conditions. There were a total of 50 and 25 replicates of inoculated and non-inoculated plants, respectively, which were arranged in a completely randomized block design in five blocks within the experimental plot . Disease incidence in plants was assessed monthly, as the proportion of plants showing disease symptoms, and severity of foliar symptoms was assessed using a 0 to 4 rating scale, according to the proportion of affected leaves and twigs  with similar ripening index (RI = 2) were combined to form a minimum of 2 kg of olive fruits per treatment and block. For this purpose, the fruits with total green color and reddish brown or black skin were discarded, so that most of the fruits had green skin with reddish spots, corresponding to RI = 2 according to Virgin olive oil was extracted using an Abencor lab plant  that mimics the industrial process of VOO production . Fruit wp-hydroxyphenyl-acetic acid (46.4 μg mL−1) and o-coumaric acid (9.6 μg mL−1) in methanol was used as internal standard, which was added (0.5 mL) to each oil sample (2.5 g) before the extraction procedure. Methanol was evaporated in a rotary vacuum evaporator at 40°C and the residue was dissolved in 6 mL of hexane. A diol-bonded phase cartridge  was conditioned by passing first 6 mL of methanol and then 6 mL of hexane. The oil solution was loaded onto the column and the solvent was pulled through. Then, the phase was washed twice with 3 mL of hexane and 4 mL of hexane/ethyl acetate . Finally, the phenolics were eluted from the cartridge with 10 mL of methanol, and the solvent was evaporated in a rotary vacuum evaporator at 40°C. The residue was dissolved in 500 μL of methanol/water , which constituted the VOO phenolics extract. Phenolics extracts were analyzed using a Beckman Coulter liquid chromatographic system equipped with a diode array detector (System Gold 168) and a Mediterranea Sea 18 column    (solvent A) and methanol/acetonitrile (50:50) (solvent B) as the mobile phases and the following elution program: (A) 0–25 min, 5–30% solvent B; (B) 25–35 min, 30–38% solvent B; 35–40 min, 38% solvent B; 40–45 min, 38–100% solvent B. Two phenolic extracts were isolated from each oil sample and duplicate analyses were carried out for each phenolic extract. Quantification of phenols and lignans was done at 280 nm except for ferulic acid, while flavones and ferulic acid quantification was carried out at 335 nm. Response factors (Rf) were used for each phenolic compound as stated by −1) = Rf × area compound/area internal standard]. Compounds were identified according to their UV–Vis spectra, and confirmed using HPLC/ESI-qTOF-HRMS with available standards on a Dionex Ultimate 3000 RS U-HPLC liquid chromatograph system , equipped with a 3-μm particle size Mediterranea Sea 18 column and operated for mass analysis using a micrOTOF-QII High Resolution Time-of-Flight mass spectrometer with qQ-TOF geometry  equipped with an electrospray ionization interface. Mass spectra were obtained in MS full scan mode. Data were processed using TargetAnalysis 1.2 software .Phenolic compounds were isolated from the oils using solid phase extraction, as described by n) after . ElutionThe extracted phenolic compounds were grouped according to the phenolic alcohol they contained (tyrosol or hydroxytyrosol), lignans, flavonoids, and phenolic acids .−1; column held for 6 min at 40°C, then programmed at 2°C min−1 to 168°C; the mass detector was operated in electronic impact mode at 70 eV; source temperature set at 230°C; and, the mass spectra scanned at 2.86 scans s−1 in the m/z 40–550 amu range. Compounds were identified by mass spectrum comparison with Wiley/NBS and NIST libraries, retention index in agreement with the literature, and co-elution with chemical standards when available. For quantification, the volatile fraction was analyzed three times on a HP-6890 GC system  equipped with the same column as previously, and operated under the following operating conditions: N2 as the carrier gas at 17 psi; injector and detector at 250°C; oven held for 6 min at 40°C and then programmed at 2°C min−1 increase to 168°C. Calibration curves were obtained using re-deodorized, high-oleic sunflower oil containing pure standards. The concentration ranges and the limits of quantification were established according to the typical volatile contents in VOO. Linear regression curves were found for all compounds, with regression coefficients greater than 0.99, and calculated Rf used for quantitation [concentration (ng/g) = Rf × area compound]. During the sample analysis, a blank that did not contain oil and a mixture of volatile standards were repeatedly run.We analyzed volatile compounds according to Extracted volatile compounds were clustered into groups according to the number of carbons in the straight-chain molecules (C6 or C5) and the polyunsaturated fatty acid of origin (LnA or LA), esters, branched-chain (BC) molecules, and terpene .1  in high-dimensional metabolomics data to produce robust and easy-to-interpret models , soil type , and the interaction between those factors. When significant differences were found mean comparisons among soil types were performed using Tukey HSD test (P = 0.05).An initial, non-supervised multivariate hierarchical clustering analysis, using Pearson’s correlation to measure distance and an average Ward clustering algorithm of all the volatile and phenolic compounds from the different treatments was performed using MetaboAnalyst 4.01 , and runt models . AdditioV. dahliae V138I isolate two months after inoculation. Symptom severity increased during the growing season, reaching a maximum in early summer. Control plants did not develop Verticillium wilt symptoms during the experiment. Maximum disease severity was observed in July, and was greater in plants grown in sandy-loam soil (severity score: 1.9) than in clay-loam (severity score: 0.92) and loam (severity score: 0.38) soils (P = 0.002) (P < 0.017) for plants grown in the sandy-loam soil (P = 0.008) olive yield, but there was no effect (P > 0.147) of type of soil or type of soil × treatment interaction: mean yield in the control was 458.7 ± 35.6 g tree−1, whereas in infected trees was 344.1 ± 28.5 g tree−1, representing a net reduction in yield of about 25% (V. dahliae treatment (32.0%) than in the control (9.3%), although this effect varied depending on the soil type  . Diseaseoam soil . Incidenoam soil . We founbout 25% . We alsoectively .V. dahliae infection on yield, we analyzed its effect on volatile and phenolic fractions that are related to the sensory quality of the VOO. In general, V. dahliae inoculation affected significantly (P < 0.05) the content of several volatile compounds, whereas the effect of soil type was less evident  affected by infection, with the dominant (E)-hex-2-enal (6C-4) being significantly increased (P = 0.001) in D-V. dahliae inoculated plants  in content of C5/LnA compounds that was from 28 to 17% of total volatiles, and affected the content of all types of compounds within this group almost equally. On average, the C5/LnA group of compounds mostly comprised pentene dimers; however, they do not appear to have any role in VOO aroma according to their odor thresholds (P < 0.001), about 50%, in D-V. dahliae infected trees, which could affect the sensory quality of VOO given its high OAV due to its extremely low detection threshold -hex-3-en-1-yl acetate (E3) may be the most important due to its higher content and lower odor threshold than other esters, such as hexyl acetate (E1) and (E)-hex-2-en-1-yl acetate (E2) (P = 0.001) values in D-V. dahliae inoculated plants as compared to control plants (Oil content of esters synthesized through the LOX pathway (LOX esters) is commercially important because they are the main contributors to oil fruitiness that are evaluated in the regulated sensory analysis for commercial grading. Among them, (ate (E2) . From thl plants .−1 oil; −1 oil; P = 0.001) values in inoculated plants  or its hydroxyl derivative hydroxytyrosol  in their molecules, lignans, flavonoids, and an array of simple phenolic acids  used in the study (V. dahliae led to oils with a significant (P = 0.007) lower content of phenolic compounds (P = 0.007) than those from the non-infected control trees, and content of the hydroxytyrosol derivatives 17% lower. In contrast, the remaining main phenolic groups in the oils (flavonoids and lignans) were unaffected by D-V. dahliae tree infection , which is likely to be the main phenolic responsible for VOO pungency (P = 0.009) in the oils from D-V. dahliae infected trees, and it is likely there would be an associated lowered perception of pungency in these oils. Similarly, content of 3,4-DHPEA-EA, which may be a key compound for bitterness in VOO (P = 0.018) in oils of infected trees. Among the oil phenolics, pungency , was 25%s in VOO , was almIn general, hierarchical clustering analysis using all volatile and phenolic compounds differentiated oils obtained from infected olive trees from those obtained from non-infected control trees . There wP < 0.01; P ≤ 0.05 with a post-hoc analysis (Fisher’s LSD) (E)-hex-2-enal (6C-4) had the highest VIP score, showing values were higher for oils obtained from D-V. dahliae infected trees. In contrast, 1-penten-3-one (5C-1) had a moderate VIP value, where content was higher in oils obtained from non-infected control plants.PLS-DA of all volatile and phenolic compounds showed a separation of the VOO obtained from infected and non-infected trees, indicating there was an altered state of metabolite levels in the VOO, as a result of tree health (r’s LSD) , and shop-HPEA-EDA and 3,4-DHPEA-EA as compounds responsible for the pungent and bitter taste notes of the oils (V. dahliae infected and non-infected trees (V. dahliae infected and non-infected trees (P < 0.001), where components 1 and 2 explained 56% of the variation in content of compounds responsible for oil flavor notes, supporting the observed results from the analysis of all compounds (p-HPEA-EDA) and the only non-desirable volatile compound  are located in the plot quadrants that correspond to the negative component 1 axis. In contrast, the area with positive component 1 is, according to the literature, more related to pleasant flavor notes and contains (E)-hex-2-enal (6C4) as well as some of the esters responsible for the fruity odor notes  and 3-methyl-butanal (BC-1). Oils obtained from trees infected with D-V. dahliae are located along the positive component 1 axis in Ocimum basilicum (sweet basil) and Mentha (mint) spp. (E)-hex-2-enal (6C4) and (Z)-hex-3-en-1-yl acetate (E-3), and the two phenolics  had lower, although moderate, VIP values.The contribution of each volatile compound to the aroma of VOO is dependent on its concentration and odor threshold. We found that only a few volatile compounds were present at levels where they contributed to oil aroma (OAV > 1) . We usedthe oils . The selthe oils . PLS-DA ed trees . We asseompounds . The loaompounds  clearly nt) spp. . PLS-DA nt) spp. , and shoV. dahliae apart from reducing olive fruit yield promoted an increase in the synthesis of C6 volatile compounds and a decrease in C5 compounds that are the main contributors to VOO aroma. Plant infection led to a reduction in the synthesis of the main phenolic compounds responsible for the taste sensory characteristics of VOO. There was a separation of oils from D-V. dahliae infected and non-infected trees, especially when considering the metabolites that really contribute to VOO flavor. To our knowledge, this is the first demonstration of the effect of Verticillium wilt infection on volatile compounds and phenolics responsible for VOO flavor. Future work integrating changes in plant metabolites combined with sensory evaluation are necessary to decipher the plant response to V. dahliae infection and the sensory quality of VOO.We found that infection of olive plants by the defoliating pathotype of The datasets generated for this study are available on request to the corresponding author.BBL and CS conceived research, performed statistical analyses, and wrote the manuscript. AGP and PL prepared materials and equipment, performed the VOO analysis, and interpreted analytical data. MM-B prepared materials, designed, and interpreted results of the field experiments. JAN-C contributed to writing the manuscript and interpreted results of the field experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD) was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB) into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described. This is owing to its unique physical and chemical properties , such as2+ , Hg2+ [12+ [2+ ,18, Pb2+ [2+ [2+  and K+ [+ [2+ [+ ,21, and + [2+ [+ , RNA [12+ [2+ [+ ,14,23,24+ [2+ [+ , cystein+ [2+ [+ , biotin + [2+ [+ , thrombi+ [2+ [+ , matrix + [2+ [+  and chol+ [2+ [+ . Despite+ [2+ [+ . The wid+ [2+ [+ . On the + [2+ [+ , indicatThe widespread use of GO as the basic platform is due to its high yielding synthesis  and its stable colloid nature in aqueous solution favored by hydrophilicity and electrostatic repulsions of the abundant oxygen functionalities . This waTo maintain the main advantages of GO in the fabrication of a graphene-based fluorescence probes, such as low cost, high yield and water solubility, it is expected that the graphene will be prepared from GO by a reduction/deoxygenation process. However, removal of the oxygen functional groups from GO surfaces via various reduction approaches to prepare reduced graphene results in an irreversible aggregation and agglomeration, and even restacking to the bulk graphite structure, which severely limits the applications of the thus-reduced graphene . This cai.e., the resulting fluorescence probe or denoted as CD-G-R. A wide spectrum of organic liquids, including water-soluble and -insoluble organic solvents, were subsequently employed as the target molecules to attack CD-G-R in order to examine the sensitivity and selectivity of CD-G-R to these molecules. To the best of our knowledge, this is the first report of the use of a graphene-based sensing platform for detection of the small organic molecules in liquid form, even though a large body of work has been dedicated to the detection of various metal ions and biomolecules using GO- or graphene-based sensing systems, as mentioned before. The present strategy for fabrication of the sensing platform CD-G-R is very simple, efficient, and cost-effective. Moreover, high fluorescence turn-on sensitivity and fair selectivity to tetrahydrofuran (THF) can be observed. THF is a polar ether and is a widely used solvent for many applications such as processing solvent, polymer production, and component in stripping fluids, cleaning fluids, functional fluids, coatings, lacquers and corrosion inhibitors [In this work, a bio-based β-cyclodextrin (β-CD) is utilized as the covalent modifier to fabricate a functionalized graphene with high water solubility by a one-pot functionalization and reduction process. The β-CD-modified and functionalized graphene will hereafter be called CD-G. The as-prepared CD-G can be used directly as the sensing platform that can quench the fluorescence dye efficiently by inclusion of the dye molecules into the CD-G and hence provide close contact of the dye with the graphene moiety, a super nanoquencher. By selectively driving the dye molecules out of the CD-G cavities based on host-guest competition interactions with given target molecules, a sensing probe can be constructed using CD-G as the basic platform. In the present work, Rhodamine B (RhB) was adopted as the fluorescence reporting molecule for CD-G to form an inclusion complex of CD-G and RhB, hibitors . A notabhibitors . Acute thibitors  and carchibitors  have beehibitors ,46 and ahibitors  have alsThe photo images and schematic illustrations given in −1 (C–O in C–OH/C–O–C) [−1 can be indexed to O–H, C–H, O–H , and C–O–C groups, respectively [The structure characterization results for GO and CD-G are shown in H/C–O–C) . This inectively . −1 (which can be attributed to the enhanced benzene ring vibrations of CD-G), suggesting the effective deoxygenation and restoration of conjugation structure of graphene. In addition, the absorption peak stemming from water becomes much smaller and blue-shifts to higher wavenumber . This is likely due to the high coverage of β-CD on the graphene surfaces, thus changing the water adsorption behavior of graphene to that of β-CD. Because of the disturbance effect of the graphene moiety on the β-CD grafted on CD-G planes, this absorption peak turns to be smaller as compared to that for pure β-CD. It is worth mentioning that the absorption corresponding to O–H group is blue-shifted from about 3,333 (for GO) and 3,399 cm−1 (for β-CD) to 3,429 cm−1 for CD-G, probably caused by the intermolecular interactions between GO and β-CD such as hydrogen bonding [d001/spacing of 0.83 nm, being much larger than that of graphite (0.34 nm) due to the incorporation of abundant oxygen functionalities) [After chemical reduction with hydrazine in the presence of β-CD, the absorptions assigned to carboxyl group, disappears completely and a new absorption band arises at approximately 1,556 cm bonding . The X-ralities) . It is nalities) . This alalities) . This isThe surface morphologies of GO and CD-G are observed by scanning electron microscopy (SEM), with the images shown in v/v). To completely quench the RhB fluorescence monitored by fluorescence spectroscopy, the mixing ratio was further increased to 35/100.The CD-G is used firstly as the nanoquencher to quench the fluorescence of the RhB solution and yield the inclusion complex of CD-G and RhB. The results are presented in Using the completely quenched inclusion complex of CD-G and RhB as the starting sensing platform, the subsequent detection of different organic liquids are performed. A range of water-soluble organic solvents was firstly detected, with the results shown in The C1-C3 monohydric alcohols, that is methanol, ethanol, and isopropanol, can turn on the fluorescence to a decent extent observable by the naked eye . The C2 dihydric (ethylene glycol) and C3 trihydric (glycerol) alcohols show a lower fluorescence switch-on efficiency as compared to the C1-C3 monohydric alcohols. This can be attributed to the fact that the increased hydroxyl groups impart higher polarity to the ethylene glycol and glycerol, which reversely reduce the competition with RhB to accommodate into CD-G because of its higher affinity to the hydrophilic outside wall (with abundant hydroxyl groups) than to the hydrophobic inner cavity of β-CD. Acetone and acetonitrile have a comparable turn-on efficiency relative to C2 and C3 monohydric alcohols, in this case ethanol and isopropanol, respectively. In addition, DMF and DMA have a similar turn-on capacity for the quenched RhB to the C1 monohydric alcohol, namely methanol (DMF shows a little higher capacity than DMA according to the fluorescence spectra). DMSO exhibits a parallel turn-on effect as compared with C2 dihydric and C3 trihydric alcohols. Moreover, THF shows an overwhelming performance over all the other examined water-soluble organic solvents, as well as over all the examined water-insoluble organic solvents . The best match with the molecular structure of inner cavity can be used to explain for the best performance of THF among the examined organic liquids since the inner surface structure of β-CD lined with the cyclic ether-like anomeric oxygen atoms and the C3-H and C5-H hydrogen atoms see  is closeTo summarize the fluorescence switch-on effect of all the organic solvent investigated in the present study, a comparison histogram of the emission intensity at about 577 nm is plotted against the mixing system with different organic liquids, with the result presented in Considering that the graphene-based sensing platform CD-G-R has a selectivity for THF among the organic liquids examined in the present study at the given excitation wavelength and scanning range. The subsequent calculation of LOD is carried out by monitoring the fluorescence spectra of CD-G-R system upon the addition of increasing concentration of THF (0–44.5 mg/mL), with the result shown in 0) for detection of THF is given as the inset of 0 and F are the fluorescence intensity in the absence and presence of THF, respectively. Error bars in the calibration curve were obtained from five parallel measurements. The pristine CD-G-R shows nearly no emission in the detected range, probably indicating that all the RhB molecules have accommodated in CD-G and thus lead to a close contact with graphene moiety and highly effective quenching of the fluorescence by FRET from RhB to the graphene. The CD-G-R showed a good linear response of the relative fluorescence intensity with respect to the THF concentration in the range of 0.218–17.5 mg/mL (r2 = 0.98816) as shown in the inset. The LOD measured as three times the signal of the blank is found to be about 1.7 μg/mL.The calibration curve of the relative fluorescence  was supplied by Oriental Chem. and Lab. Supplies Ltd. . β-cyclodextrin  was purchased from Aladdin Chemistry Co. Ltd. . Rhodamine B  was obtained from Sigma-Adrich. All other chemicals were purchased from Oriental Chem. and Lab. Supplies Ltd.  and used as received.Graphite fine powder (E.P.) was obtained from Tianheng Technology Co. Ltd. . Hydrazine hydrate of A.R grade (N3 (1.0 g) were placed in a flask (250 mL), followed by addition of concentrated H2SO4 (50 mL) in small portions under magnetic stirring and cooling by an ice bath. The mixture was kept stirring at 5 °C for 2 h, and then KMnO4 (7.3 g) was added portionwise under cooling and continuously stirring conditions to maintain the temperature of the suspension lower than 20 °C. Afterwards, the temperature was increased to 35 °C, and the reaction mixture was stirred continuously at this temperature for 30 min. DI water (90 mL) was then added gradually, and the mixture was stirred for another 15 min. Additional DI water (55 mL) was added, followed by slow addition of H2O2  for the purpose of reducing the residual permanganate to soluble manganese ions. Finally, the mixture was filtered and washed thoroughly with diluted HCl , DI water (150 mL) and methanol (150 mL), and then vacuum-dried at 50 °C for 24 h.GO was synthesized by a modified Hummers method as described elsewhere . BrieflyAs-prepared GO powder (30.0 mg) and DI water (60.0 mL) were placed in a beaker, followed by sonication treatment for 1 h, which yielded a homogeneous yellow-brown suspension of graphene oxide (0.5 mg/mL). Separately, β-cyclodextrin (1.6 g) was added to a flask loaded with DI water (40.0 mL), and then magnetically stirred at 50 °C to dissolve the β-cyclodextrin. Afterwards, a portion of graphene oxide suspension (24 mL), ammonia (0.8 mL) and hydrazine hydrate (25.0 μL) were homogenized into the β-cyclodextrin solution, followed by a reaction at reflux under constant stirring at 80 °C for 3.5 h, leading to a black dispersion. The as-formed dispersion was charged into a dialysis bag for dialysis treatment against flowing DI water for 3 days, further washed repeatedly with DI water (50 mL) followed by ethanol (50 mL), vacuum-filtered using a nylon membrane with 0.22 um pore size, and finally dried under vacuum at 50 °C for 24 h. The resulting black powder was stored in a desiccator prior to use.The as-synthesized CD-G powder (26.5 mg) was homogenously dispersed into DI water (100 mL) by ultrasonication for 1 h. The obtained CD-G dispersion (0.265 mg/mL) was employed as the fluorescence nanoquencher. The fluorescence solution of RhB (50 nM) was prepared by dissolving the fluorescence dye RhB (0.012 mg) in DI water (500 mL). The CD-G dispersion (0.265 mg/mL) was mixed into the RhB solution  in small portions by mild stirring each for 3 min to allow the sufficient interactions between RhB molecules and CD-G sheets. The gradual quenching process can allow monitoring of the RhB fluorescence quenching behavior by taking the digital images and recording the fluorescent spectra. The final inclusion complex of CD-G and RhB, with completely quenched fluorescence determined by the fluorescence spectroscopy, was used as the fluorescence switch-on probe for detection of a wide range of organic liquids.2 atmosphere at a heating rate of 10 °C/min. The surface morphologies were observed by a field emission scanning electron microscopy . TEM images and SAED patterns were obtained by a Jeol JEM-2011 TEM facility at an acceleration voltage of 100 kV. The specimens for the TEM observation were prepared by dropping a diluted ethanol dispersion of the GO and graphene specimens onto a silicon wafer, followed by drying at room temperature. Fluorescence spectra were recorded on a Perkin-Elmer Luminescence spectrometer (LS50B), which was equipped with a motor-driven linear polarizer on the detection side, at room temperature under isotropic excitation.UV/Vis spectra were measured on a Lambda 18 UV/Vis spectrometer. FTIR spectra were recorded on a FTIR spectrometer (PerkinElmer System 2000) in KBr pellet mode. The powder XRD patterns were collected by a Bruker D8 Advance X-ray diffractometer . TGA tests were performed on a Mettler Toledo TGA/SDTA851 under NAn organic liquids-responsive graphene-based fluorescence probe has been fabricated in the present study by a simple, efficient and cost-effective method. To fabricate the probe, a one-pot chemical reduction and functionalization of GO with hydrazine in the presence of β-CD has been firstly conducted, which leads to the β-CD-modified and functionalized graphene. The β-CD has been demonstrated to be grafted on the graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as UV/Vis, FTIR, XRD TGA, SEM and TEM. The subsequent fabrication of the fluorescence probe has been achieved by inclusion of RhB molecules into CD-G using a facile and economical solution mixing method. The RhB fluorescence can be quenched controllably based on FRET from RhB to graphene through changing the mixing ratio of CD-G to RhB. A wide range of organic solvents including water-soluble and -insoluble ones has been tested using the fluorescence probe, which shows the highest sensitivity to THF, with the LOD of about 1.7 μg/mL. Some insight into the mechanism for explaining the different fluorescence responses of the probe to the examined targets has been provided. This work thereby shows a great significance for sensing and detection applications in a relatively unexplored field of detecting organic liquids using graphene-based platform, especially for the waste water containing THF contaminant, e.g., solvent leakage detection of tanks and piping systems. Based on the fluorescence spectroscopy, quantitative analysis of organic solvent concentration in water can also be realized using our developed graphene-based sensor. This work on the fabrication of the label-free graphene-based fluorescence probe by a simple, efficient and cost-effective strategy has opened up new avenues for development of other effective graphene-based probes for various sensing, monitoring and detection applications.


Competing risks occur when populations may experience outcomes that either preclude or alter the probability of experiencing the main study outcome(s). Many standard survival analysis methods do not account for competing risks. We used mortality risk in people with diabetes with and without albuminuria as a case study to investigate the impact of competing risks on measures of absolute and relative risk.A population with type 2 diabetes was identified in Clinical Practice Research Datalink as part of a historical cohort study. Patients were followed for up to 9 years. To quantify differences in absolute risk estimates of cardiovascular and cancer, mortality standard (Kaplan-Meier) estimates were compared to competing-risks-adjusted (cumulative incidence competing risk) estimates. To quantify differences in measures of association, regression coefficients for the effect of albuminuria on the relative hazard of each outcome were compared between standard cause-specific hazard (CSH) models  and two competing risk models: the unstratified Lunn-McNeil model, which estimates CSH, and the Fine-Gray model, which estimates subdistribution hazard (SDH).In patients with normoalbuminuria, standard and competing-risks-adjusted estimates for cardiovascular mortality were 11.1%  10.8–11.5%) and 10.2% (95% CI 9.9–10.5%), respectively. For cancer mortality, these figures were 8.0% (95% CI 7.7–8.3%) and 7.2% (95% CI 6.9–7.5%). In patients with albuminuria, standard and competing-risks-adjusted estimates for cardiovascular mortality were 21.8% (95% CI 20.9–22.7%) and 18.5% (95% CI 17.8–19.3%), respectively. For cancer mortality, these figures were 10.7% (95% CI 10.0–11.5%) and 8.6% (8.1–9.2%). For the effect of albuminuria on cardiovascular mortality, regression coefficient values from multivariable standard CSH, competing risks CSH, and competing risks SDH models were 0.557 (95% CI 0.491–0.623), 0.561 (95% CI 0.494–0.628), and 0.456 (95% CI 0.389–0.523), respectively. For the effect of albuminuria on cancer mortality, these values were 0.237 (95% CI 0.148–0.326), 0.244 (95% CI 0.154–0.333), and 0.102 (95% CI 0.012–0.192), respectively.Studies of absolute risk should use methods that adjust for competing risks to avoid over-stating risk, such as the CICR estimator. Studies of relative risk should consider carefully which measure of association is most appropriate for the research question.The online version of this article (10.1186/s41512-018-0035-4) contains supplementary material, which is available to authorized users. In prognostic studies, time-to-event data may be incomplete for many subjects as a result of loss to follow-up, reaching the end of the study, or withdrawal from the study. The truncation of subject follow-up resulting from incomplete data is typically referred to as ‘censoring’. Most standard survival models, like the Kaplan-Meier estimator  and Cox Patients are seldom at risk of experiencing only a single type of outcome.Competing risks are defined as events during follow-up that either preclude the observation of the primary outcome, or alter the probability of its occurrence . The effRoughly 3.5 million people are currently living with diabetes mellitus in the UK, of which 90% have type 2 diabetes mellitus (T2DM) . T2DM isT2DM and albuminuria have both been independently associated with an increased risk of mortality from cardiovascular disease , 15 and Using the impact of albuminuria on competing mortality in T2DM as a case study, we set out to evaluate the effect of implementing competing-risks-adjusted methods on measures of absolute risk and measures of association.The Clinical Practice Research Datalink (CPRD) is the world’s largest database of anonymised primary care data, spanning 674 practices in the UK . The datPatient eligibility was defined using only data uploaded by GP practices after the date they were classified as up-to-standard. Patients were required to be aged 35 years or over with T2DM on the study start date, have gender unambiguously recorded, have CPRD records that could be linked to Office for National Statistics data and have at least 2 years or uninterrupted follow-up data from their current registered general practice before the study start date for the evaluation of baseline risk factors. The presence of T2DM was established using a combination of medical codes, product codes and patient age.Patients were excluded whose records contained medical codes for other forms of diabetes, polycystic ovary syndrome, total pancreatectomy or pancreatic or renal transplant, at any point prior to the study start date.Follow-up commenced on the study start date of 1 January 2005 and terminated on the study end date of 1 January 2014. Patient follow-up ceased at the earliest event of the study end date, the date of patient mortality, the date of last data collection from the patient’s practice, the date the patient transferred out of their current practice and the date the patient underwent total pancreatectomy, or pancreatic or renal transplant.Albuminuria status was defined using definitions present in the 2013 Kidney Disease Improving Global Outcomes guidelines . Within 1c) and total-to-high-density-lipoprotein  cholesterol ratio.Adjustment variable data were extracted from CPRD on patient: age, gender, body mass index (BMI), smoking status, systolic blood pressure (SBP), glycated haemoglobin  codes for the ‘underlying cause’ of death listed on UK Office for National Statistics death certificates. Within these, cardiovascular mortality was defined as death with ICD-10 codes I10-I79, cancer mortality was defined as death with ICD-10 codes C00-C97 and other mortality as death with ICD-10 codes other than those listed for cardiovascular or cancer mortality.t tests , interest . Competit is an estimate of the risk of failure from a specific cause, acknowledging that the absolute risk of the event is lowered by the presence of other competing risks. Individuals are removed from the at-risk set, at any instant, only if they have previously experienced the primary event or any censoring events that are explicitly assumed to be non-informative but retained in the at-risk set if they have experienced a competing outcome [The cumulative incidence competing risk (CICR)  method e outcome .The Cox proportional hazards model  estimateWhen competing risks are present, the different risk sets employed by cause-specific hazard models (like the Cox-PH or Lunn-McNeil model) and subdistribution hazard models (like the Fine-Gray model) give rise to different measures of association. The cause-specific hazard ratio may be thought of as a measure of ‘aetiological association’, i.e. best suited to quantifying causal relationships. Conversely, the subdistribution hazard ratio may be thought of as a measure of ‘prognostic association’, i.e. best suited to quantifying predictive relationships .The proportional cause-specific hazard assumptions of the Cox-PH and Lunn-McNeil models are assessed using Schoenfeld residuals . SchoenfSensitivity analyses were conducted to assess the robustness of all risk estimates to the misclassification of cause of mortality on patient death certificates. Within these analyses, mortality was reclassified as being attributable to either cardiovascular disease or cancer if the competing cause was listed as a contributing factor. The potential misspecification of the study primary exposure was assessed by re-assigning patient baseline albuminuria status using only read codes, numerical test values or categorical test values. Additionally, the robustness of estimates of absolute risk was evaluated through the use of an alternate cumulative risk estimator , while the robustness of estimates of relative risk was evaluated through the inclusion of time-interaction terms to correct for violations of the proportionality assumption, and the use of alternate parameterisations of the Lunn-McNeil model, in which competing mortality was restructured into a single outcome, representing all mortality not attributable to the primary outcome.Of the 86,962 patients eligible for inclusion, it was possible to ascertain the baseline presence/absence of albuminuria in 54,801; 42,662 without albuminuria and 12,139 with albuminuria. Patients with albuminuria at baseline were older, had higher systolic blood pressure, had worse glycaemic control and were more likely to be current or ex-smokers. No significant associations were found between baseline albuminuria and gender, body mass index or total-to-high-density-lipoprotein cholesterol ratio. Over the course of 9 years of follow-up (median 7.7 years), 14,201 patients died: 9558 (67.3%) with normoalbuminuria at baseline and 4643 (32.7%) with albuminuria at baseline. Of these 14,201 patients, 5574 (39.3%) died from cardiovascular causes, 3455 (24.3%) died from cancer and 5172 (36.4%) died from other causes. Further detail pertaining to the entire study cohort is presented in Table p < 0.001) were found between the survival functions and cumulative incidence functions of the albuminuria strata for both cardiovascular and cancer mortality.Among 5574 patients who died from cardiovascular causes, 3661 (65.7%) had normoalbuminuria at baseline and 1913 (34.3%) had albuminuria at baseline. Among 3455 patients who died from cancer, 2564 (74.2%) had normoalbuminuria at baseline and 891 (25.8%) had albuminuria at baseline. Among 5172 patients who died from causes other than cardiovascular disease or cancer, 3330 (64.4%) had normoalbuminuria at baseline and 1842 (35.6%) had albuminuria at baseline. Statistically significant differences  was larger in variable-unadjusted models and larger for the outcome of cardiovascular mortality. For a given outcome, model estimates from the Cox-PH and Lunn-McNeil cause-specific hazard models were similar, whereas model estimates from the subdistribution hazard Fine-Gray model were lower. Regardless of these trends, model estimates within respective outcomes did not differ to a degree that would have resulted in drastically different clinical implications had the incorrect measure of risk been modelled.Albuminuria was found to be significantly associated with the cause-specific hazards and subdistribution hazards of cardiovascular and cancer mortality in both univariable and multivariable analyses . Strengths of the CPRD dataset include its size and its representativeness of the UK . LimitatThis is a single case study of competing risk methodology, limited to methods identified as being commonly used in the literature and readily implementable in statistical packages. Thus, the results described may not mirror the findings in other clinical areas, or from different models.Our findings for absolute risk estimates are consistent with those from studies of cancer progression in patients with head or neck cancer , cancer Several authors have previously compared Fine-Gray to Cox models , 36–44. We have not addressed here the stratified Lunn-McNeil model, because it is known to give near-identical results to the Cox model . A previStudies of absolute risk should use methods that adjust for competing risks to avoid over-stating risk, such as the CICR estimator. Studies of relative risk should consider carefully which measure of association is most appropriate for the research question. Cause-specific hazard ratios will be appropriate when the aim of the study is to establish a potentially causal relationship between an exposure and an outcome. Clinical trials, for example, are primarily interested in the relationship between allocated treatment group and the primary outcome. In these cases where cause-specific hazard ratios are appropriate, we find, in common with previous authors, little difference between estimates from the Cox model and from the Lunn-McNeil competing risk model. Subdistribution hazard ratios will be appropriate when the aim of the study is to quantify burden of disease, for example, in combination with a CICR estimate to produce a prognostic model. As noted above, subdistribution hazard ratios have the property that a beneficial effect of the exposure on a competing outcome may appear as a harmful effect on the outcome of interest, or vice versa.Our findings, the previous literature and common sense confirm that researchers should consider competing risks when selecting statistical analysis methods. The widely used Kaplan-Meier method arguably over-estimates absolute risk, sometimes substantially, and though the widely used Cox proportional hazards method for relative risk will often be a reasonable approximation, users should at least be making an informed choice to use cause-specific rather than subdistribution hazard methods.Additional file 1:Table S1. Adjusted estimates for the effect of baseline risk factors on cardiovascular mortality from Cox-PH, Lunn-McNeil and Fine-Gray Models. (DOCX 15 kb)Additional file 2:Table S2. Adjusted estimates for the effect of baseline risk factors on cancer mortality from Cox-PH, Lunn-McNeil and Fine-Gray Models. (DOCX 15 kb)Additional file 3:Figure S1. Proportional cause-specific hazards assessment cardiovascular mortality. (PDF 1874 kb)Additional file 4:Figure S2. Proportional cause-specific hazards assessment for cancer mortality. (PDF 1233 kb)Additional file 5:Figure S3. Proportional cause-specific hazards assessment for other mortality. (PDF 1695 kb)Additional file 6:Figure S4. Proportional subdistribution hazards assessment for cardiovascular mortality. (PDF 1100 kb)Additional file 7:Figure S5. Proportional subdistribution hazards assessment for cancer mortality. (PDF 865 kb)